• I never trust primers from Papers for qPCR especially if they are 5 years or more old
• Accordingly, as you are new to the lab and I have been performing qPCR and PCR for 25 years, find attached a stratagene general qPCR guide and also a couple of SOPs I put together on general primer design and retrieving Ref Sequences from data bases (Ensembl and GenBank) to make primers for qPCR
• Read those documents - especially the SOPs and get back to me with questions
• In summary though there are salient points pertaining to primer design for qPCR; Read the SOPs on transcript (Ref Seq) retrieval from gen bank to apprehend details:
• Put your full gene name (GO) into Genbank
• https://www.ncbi.nlm.nih.gov/gene
• Retrieve just those transcripts (Ref Seq) of your gene that have been empirically verified by PCR
• These are denoted by the Prefix 'NM' and not things like NX which are predicted based on gene annotation but not actually proven to exist
• Now cross Reference those transcripts with Ensembl:
• https://www.ensembl.org/index.html
• Just focus on those transcripts independently verified by both data bases
• Import the last 2kb of mRNA (ref) Sequence into Primer BLAST:
• https://www.ncbi.nlm.nih.gov/tools/primer-blast/
• Tell the software to design primers to exon-exon boundaries (this will ensure you qPCR primers can only target mRNA with contiguous exons and not gDNA; primers internal to exons can bind both mRNA and gDNA
• Alternatively, retrieve your transcript (Ref seq) and import into IDT primer quest and this will design primers suitable for qPCR:
• https://eu.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex
• You will need to set up an account to Access the IDT Primer quest qPCR primer design tool
• Finally, take the most 3' primers it suggests and subject them to BLAST to make sure
• https://www.ncbi.nlm.nih.gov/tools/primer-blast/

• If exon exon boundary primers are only avaialble more than 2kb from your Poly A tail then you can situate your primer within a common exon within 3' biased exons but just make sure you have digested away all of your genomic DNA (this is good practice regardless)

Hello,

First, any primers used by the various individual looking for mutation can be used as they are specific for wild type TP53. If you need them, contact me by email. Nevertheless, I will be very cautious before starting this project and put more thoughts. Sarcoma (and more often osteosarcoma) is the only tumour type where you can find deletion of the TP53 gene. Many of these deletions target the 5' end of the gene but other car encompass the whole gene. Therefore, I think that using a single primer is not sufficient and you should use another methodology such as MLPA. It will be very important to know the contamination of your tumor by normal cells.

Best

Thierry

• I never trust primers from Papers for qPCR especially if they are 5 years or more old
• Accordingly, as you are new to the lab and I have been performing qPCR and PCR for 25 years, find attached a stratagene general qPCR guide and also a couple of SOPs I put together on general primer design and retrieving Ref Sequences from data bases (Ensembl and GenBank) to make primers for qPCR
• Read those documents - especially the SOPs and get back to me with questions
• In summary though there are salient points pertaining to primer design for qPCR; Read the SOPs on transcript (Ref Seq) retrieval from gen bank to apprehend details:
• Put your full gene name (GO) into Genbank
• https://www.ncbi.nlm.nih.gov/gene
• Retrieve just those transcripts (Ref Seq) of your gene that have been empirically verified by PCR
• These are denoted by the Prefix 'NM' and not things like NX which are predicted based on gene annotation but not actually proven to exist
• Now cross Reference those transcripts with Ensembl:
• https://www.ensembl.org/index.html
• Just focus on those transcripts independently verified by both data bases
• Import the last 2kb of mRNA (ref) Sequence into Primer BLAST:
• https://www.ncbi.nlm.nih.gov/tools/primer-blast/
• Tell the software to design primers to exon-exon boundaries (this will ensure you qPCR primers can only target mRNA with contiguous exons and not gDNA; primers internal to exons can bind both mRNA and gDNA
• Alternatively, retrieve your transcript (Ref seq) and import into IDT primer quest and this will design primers suitable for qPCR:
• https://eu.idtdna.com/site/account/login?returnurl=%2FPrimerquest%2FHome%2FIndex
• You will need to set up an account to Access the IDT Primer quest qPCR primer design tool
• Finally, take the most 3' primers it suggests and subject them to BLAST to make sure
• https://www.ncbi.nlm.nih.gov/tools/primer-blast/

• If exon exon boundary primers are only avaialble more than 2kb from your Poly A tail then you can situate your primer within a common exon within 3' biased exons but just make sure you have digested away all of your genomic DNA (this is good practice regardless)

Hi Laurence

I am new to the lab also. I hope you don't mind if I download your docs also?

Jacinta

Of course

If you look on my methods you will find alot of other docs

Help yourself